TGC method for inducting targeted somatic transgenesis

ABSTRACT

The present invention concerns a TGC method for inducting targeted somatic transgenesis in an animal host, whereby bacteria with a foreign DNA integrated into an episomal vector release, under the control of eukaryotic regulatory elements forulterior transcription and expression, said foreign DNA in the case of infection of a foreign organism, organ, tissue, cell line or individual cells, causing transcription and expression of foreign DNA and/or foreign protein in said location.

The object of the invention is a method for inducting targeted somatictransgenesis (TGC=targeted genetic conditioning), which is used forexpressing foreign proteins in cells, tissue, organ or an entire hostorganism, as well as for somatic gene therapy.

It is known that proteins for technical application or for therapeuticpurposes can be expressed in sufficient quantity by the transfer ofgenes in microorganisms or mammalian cells. These procedures areparticularly important for proteins occurring naturally in the body,such as hormones, regulatory factors, enzymes, enzyme inhibitors andhumanized monoclonal antibodies which are otherwise only available to alimited extent or not available at all. The procedures are alsoimportant for producing surface proteins of pathogenic microorganisms orviral envelope proteins so as to safely produce diagnostic tests and forthe development of efficacious vaccines. Through protein engineering itis also possible to produce new types of proteins, which through fusion,mutation or deletion of the corresponding DNA sequences, have propertiesoptimized for particular uses, for example immunotoxins.

Genes obtained from human cells are also functional in mouse, rat orsheep cells and there lead to the formation of corresponding geneproducts. This has already been made use of in the production oftherapeutic products, for example in the milk of transgenic farmanimals. The hitherto known method has been by the microinjection ofcorresponding foreign DNA carrying vectors into the nucleus of thefertilized egg cell, in which the DNA is then incorporated into thechromosome with a yield of 1%. The transgenic fertilized egg cell isthen transplanted into hormonally stimulated mother animals. Anoffspring carrying the transfected gene in all its body cells is thebasis for the creation of a “transgenic herd/flock”. Using genetechnology it is now possible to alter farm animals in such a targetedway that they produce human proteins in their blood, tissue or milk,which cannot be produced by microorganisms or plants.

However, the use of transgenic animals as protein production factorieshas the decisive disadvantage that it is necessary to manipulate thegerm line of the animal. Due to the considerable expenditure oftechnology and time required to create and breed transgenic animals andalso due to the discussions regarding the ethical consequences of thesemethods, alternative methods for producing proteins in animal hostswithout manipulation of the germ line are necessary and would be veryadvantageous.

It is known, furthermore, that the milk of mammals such as cows, sheep,goats, horses or pigs can contain a range of disease-causing bacterialagents. Among such agents are Listeria, Mycobacteria, Brucella,Rhodococcus, Salmonella, Shigella, Escherichia, Aeromonads and Yersiniaor general bacteria with intracellular lifestyle [1, 2]. These bacteriaare usually transmitted to humans or animals through oral ingestion [3],but can also be transmitted by droplet infection. A major source for theinfection of humans with Listeria [4], Mycobacteria [5] and Escherichiacoli is contaminated milk [6]. Humans ingest the bacteria when consumingunpasteurised milk or milk products. The other bacteria types listedabove, such as Salmonella, Shigella, Yersinia, Rhodococcus and Brucellaare transmitted to humans in a similar way. However, bacteria may alsoenter humans through other bacterially infected animal products fromcows, goats, sheep, hares, horses, pigs or poultry.

The infection of animals frequently occurs through mucosal surfaces andvery frequently through the digestive tract. However, after ingestion ofbacteria, for example in the case of Listeria, not all tissues showsymptoms of infection. In cows and goats the infection is mainly evidentin the udder, spleen and liver. In sheep there may additionally beillness in the central nervous system in the form of meningitis, so notall animals survive the infection. With infection of the udder, theinfection chain is closed. With contaminated milk, acquired bacteria canreinfect another animal, for example a suckling calf or a human via thedigestive tract.

The following is known at present regarding the process of bacterialinfection in humans, here presented using the example of Listeria:

Of the six known Listeria species, only L. monocytogenes and L. ivanoviiare pathogenic for humans [7]. Illness in humans results from consuminginfected milk or milk products. The course of the illness depends on thestate of health of the individual and is generally inapparent.Intrauterine transmission of bacteria to the fetus may occur duringpregnancy, resulting in abortion, stillbirth or premature birth. In allcases excellent and problem-free treatment exists using antibiotics suchas ampicillin or erythromycin [8; 8a].

The mode of entry into the cell occurs is well defined for L.monocytogenes in humans and animals and for L. ivanovii in sheep. Forfull pathogenicity of Listeria to occur, a range of pathogenicityfactors are necessary. Among them are PrfA (positive regulator ofvirulence), ActA (actin nucleating protein), PlcA(phosphatidylinositol-specific phopholipase), PlcB(phosphatidylcholine-specific phopholipase), Hly (listeriolysin), Mpl(metalloprotease) [9]. The cell specificity of the pathogen—host cellinteraction is mediated through a range of proteins. Among these are theinternalins InlA and InlB, which are involved in the initial contact andthe interaction of bacteria and cell surface [10, 11]. Underexperimental conditions L. monocytogenes can also infect endothelialcells, epithelial cells, fibroblasts and hepatocytes. In addition, L.monocytogenes can infect cells of the white blood cell count likeneutrophilic granulocytes, macrophages and lymphocytes. This is asignificant factor in the transmission of bacteria from the site ofprimary infection to the target organ in the host. Finally, lung tissuecan also be infected by Listeria if the bacteria are applied as adroplet infection.

After adhering to the cell surface, L. monocytogenes is taken up by thecell by endocytosis, the bacterium breaks down the endosome membraneunder the effect of listeriolysin (Hly) and is thus released into thecell cytosol [14]. Once inside the cell, the bacteria can proliferate.With the production of further proteins, the fully pathogenic bacteriumdoes not stay localized but actively spreads to distal sites. Bacterialspread is effected by using a range of proteins from L. monocytogenesitself and some cellular proteins [15, 16]. ActA is expressed on thecell surface of L. monocytogenes. It binds the cellular protein VASP,which for its part forms the bridge required for the attachment ofcellular actin. Actin tails subsequently develop which carry thebacterium at their tip and thus move it further through the cell. If L.monocytogenes contacts the cell membrane, a membrane protrusion forms,which projects directly into any adjacent cells if they are present.This protrusion is then endocytosed by the adjacent cell so the L.monocytogenes is then inside the new cell within a double membrane. Thetwo membranes are dissolved under the effect of Hly and PlcB [17]. Atthe end of this process L. monocytogenes has also infected theneighbouring cell and the infection process begins again. In this way L.monocytogenes enters, for example, secretory cells of the cow udder.Secreted Listeria proteins are detectable in milk, i.e. they are passedon intracellularly from the lactating cell into the milk [18]. Hly(listeriolysin) and IrpA (internalin related protein [19]) are twopathogenicity factors belonging to this group of proteins which areproduced, secreted and passed out in milk in large quantities by L.monocytogenes [20].

Knowledge of the infection process has made it possible to alter L.monocytogenes genetically in such a way that it expresses foreignproteins. Examples for the expression of foreign proteins in L.monocytogenes are: alkaline phosphatase from Escherichia coli,nucleoprotein from influenza virus, major capsid protein (L1) fromcottontail rabbit papillomavirus (CRPV) and Gag protein from HIV type 1[20 to 27].

In addition to proteins of prokaryotic origin, this also applies toviral proteins which are not normally produced within eukaryotic cells.These viral proteins and similar foreign proteins of prokaryotic andeukaryotic origin can be produced by L. monocytogenes without aeukaryotic cell being needed. Proteins produced by L. monocytogenes aresecreted into the milk.

Infection by bacteria occurs through specific interactions of ligandproteins of the bacteria with receptor proteins of the target cells. Inthe case of L. monocytogenes, the internalin family plays a significantrole; the internalin proteins determine to a large extent the cellspecificity of the infection process [28]. Additionally, an ActAdependent cell ingestion has been discussed, which is mediated throughreceptors of the heparan sulphate family [29]. If L. monocytogenesinfects a cell, it does not lead to a full infection cycle in everycase. If listeriolysin in L. monocytogenes is inactivated, the bacteriathen remain in the endosome and the infection in the “first cell”doesnot take place. Bacteria in which the protein ActA is deleted, inactiveor no longer available, enter the first infected cell but remain thereand can no longer infect the neighbouring cells [30, 31]. If PclB isdeleted, the bacteria are no longer able to establish itself in thesecond cell.

L. monocytogenes is a bacterium which can be treated with a range ofantibiotics. Ampicillin and penicillin (always in combination withgentamycin) are particularly suitable. Erythromycin and sulphonamidescan also be used as alternatives. Tetracycline, vancomycin orchloramphenicol can also be used in special cases [32]. Similartreatments exist for other bacteria [8a] of the following types:Aeromonads, Bartonella, Brucella, Campylobacter, Enterobacteriaceae,Mycobacterium, Renibacterium, Rhodococcus and other bacteria which aregenetically or biochemically related to them.

Given this information, the question arises as to how bacterialinfection can be used to induce organotropic protein production.

This problem is solved by a TGC procedure that induces targeted somatictransgenesis, whereby bacteria, carrying a foreign DNA which isintegrated into an episomal vector and prepared for subsequenttranscription and expression, release their genetic information into aninfected single cell when infecting cells, tissue, an organ or the wholehost organism and so cause expression of the foreign protein.

This method can be used to obtain a foreign protein but is alsoadvantageous for somatic gene therapy. Here the foreign DNA, introducedinto the host organism through bacterial infection, can cause theproduction of protein missing in the host organism or, by producingsingle or double strand nucleic acids, can increase, reduce or hinderthe production of a protein in the host organism. This method can beused on all known farm animals and also on humans.

If the infected tissue is the egg of a poultry bird, the foreign proteinis produced in the egg and can be isolated following known proceduresfor the isolation of proteins, for example from hen eggs. If theinfected tissue is blood cell tissue, the bacteria can spread viaparenteral infection of the cells and through them the foreign DNA canreach the entire infected organism. If the host animals are laboratoryanimals whose infected organ is an udder, the desired foreign protein isthen produced in the milk of the laboratory animal from which theforeign protein can then be isolated.

The TGC procedure is discussed below using the L. monocytogenesbacterium as an example. It can be similarly used, however, for allbacteria which grow intracellularly, in particular bacteria of thefollowing types: Aeromonads, Bartonella, Brucella, Campylobacter,Clostridia, Enterobacteriaceae (in the case of the latter, particularlybacteria of the genus Yersinia, Escherichia, Shigella, Salmonella),Legionella, Mycobacterium, Renibacterium, Rhodococcus and bacteria fromgenetically or biochemically related types. Other bacteria types whichare non-pathogenic and do not have an intracellular lifestyle are alsosuited to the method according to the invention, as long as they areviable in a eukaryotic host organism.

It is additionally possible to carry out the TGC procedure withnaturally apathogenic bacteria which through genetic manipulation arearmed with additional factors which enable their entry into cells. Manynaturally occuring bacteria such as Bacillus subtilis, Lactobacilli,Pseudomonads, Staphylococcus incapable of intracellular growth can beadditionally equipped with a set of pathogenicity factors, for thispurpose. One TGC safety strain armed in this way is, for example,Bacillus subtilis, which is additionally equipped with listeriolysinfrom L. monocytogenes. An example for the arming of apathogenic bacteriafor the TGC safety strain is given in example 1, with the equipping ofL. innocua with the hly and/or actA gene from L. monocytogenes. Afurther example is E. coli K12 armed with the invasin gene (inv) fromYersinia pseudotuberculosis.

The TGC procedure is carried out in the following steps:

a) Cloning of the TGC (foreign) DNA:

The TGC method is initiated with the preparation of L. monocytogenesstrain in the laboratory. The cDNA for the foreign protein to beproduced is inserted into a suitable vector. The introduction of thecDNA is carried out in a known way so that subsequent transcription andexpression in the eukaryotic host is assured. If the protein is secretedfrom the cell then the vectors must contain suitable host cell specificsecretory signal sequences. The vector can be a eukaryotic vector, forexample pCMV from the company Clontech or PCMD from the companyInvitrogen, both of which are commercially available. As importantcriteria for chosen vectors, these have eukaryotic promoters, donors andacceptor sites for RNA splicing (optional property), as well as apolyadenylating site, for example from SV40. The production of geneticconstructs (hereafter referred to as TGC DNA below) in E. coli, or anyother suitable host strain according to the method, can be carried outfor the propagation of the DNA. The TGC DNA must simply be able to beintroduced into the selected bacteria for the primary cloning and thenlater transferred into the selected bacterial TGC safety strain. Thetransfer into L. monocytogenes can be carried out using the variouswell-known methods of gene transfer of isolated DNA (transformation,electroporation etc.) or can be undertaken using the processes ofconjugation and transduction either directly or indirectly frombacterium to bacterium.

b) TGC safety strains as recipients of TGC DNA:

Special L. monocytogenes host strains are used as recipients of the TGCDNA,—or other TGC hosts, which like L. monocytogenes are intracellularlyactive bacteria (e.g. Yersinia) or bacteria which enter the endosome(e.g. Salmonella) or are “armed” with additional bacterial factors, oralternatively, otherwise non-pathogenic bacteria (e.g. Escherichia colior L. innoca). In all these cases the following properties, singly or incombination, must be met:

-   (A.1) they are suitable as recipients of foreign DNA (genetic    manipulability);-   (B.1) they carry mutations which affect genes, without which    survival of the bacteria in the environment (outside the host) is    not possible, for example, at low ambient temperatures (safety    related property);-   (B.2) they are attenuated host strains, for which a part of their    virulence factors are deleted or inactivated so that they no longer    possess the full pathogenicity of the wild-type strains    (attenuation);-   (C.1) they are “genetically disabled” and can only be cultivated on    defined artificial media due to targeted metabolic defects    introduced by the experimenter. As a result of these defects they    are incapable of growth in a cell and in particular in the animal    host and thus cannot proliferate and undergo “endogenous suicide”;-   (C.2) they induce their uptake in endosomes and are dissoluted in    these cell compartments (infection via endosomes);-   (C.3) they are ingested by professional phagocytes but can dissolve    these cell compartments (i.e. egress) (infection through    phagolysosomes);-   (C.4) the bacteria carry suicide genes which are only conditionally    activated after invading the host cell, so the bacteria kill    themselves (“exogenous suicide”);-   (D.1) they can be eliminated by antibiotic treatment of the intended    animal host (killing off through antibiosis).

Point A.1 is a general property of bacteria, without which none of thegenetic manipulation mentioned would be possible.

Points B.1 and B.2 summarize alterations which make the use of thebacteria safer. Bacteria with these alterations cannot proliferate ifreleased to the outside world, are attenuated (B.1), or show reducedpathogenic potential (B.2). The alteration of bacteria according topoint B.1 has an influence on the release of foreign DNA into the cell(see points C.2 and C.3).

Points C.1-C.4 refer to genetic alterations of bacteria which decisivelydetermine the release of the foreign DNA into the animal cell. In pointsC.3-C.4 are indicated ways of infection which for bacteria, furthersummarized below in the examples, were identified as a means for thetransmission of foreign DNA into the cytosol of animal cells.

Antibiotic treatment carried out in point (D.1) permits the targeteddestruction of bacteria. As a result of this, foreign DNA is releasedfrom the bacteria and therapy with antibiotics is also a safety relevantfeature.

The alterations and interventions of C.1-C.4 and also B.2 and D.1 enablethe release of recombinant DNA into the cell.

Strains with these properties (singly or in combination) are called TGCsafety strains.

c) Optimization of the TGC hostss to the target organ of the TGCprocedure:

The TGC DNA which codes for the foreign protein to be produced istransferred into the TGC safety strain by transformation, conjugation ortransduction. The strains thus obtained are subsequently referred to asTGC hosts. The host supplies (feeds) the TGC host with DNA and therebyinduces somatic transgenesis. In order for the desired foreign proteinto be optimally expressed during the TGC process, the gene should bepreferably controlled by promoters and other regulatory sequences thateither originate from the preselected target organ of the TGC process orare optimized for the target organ, as for example with udder specificpromoters and secretion signals.

d) Infection of the host organism with the TGC host:

The propagation of the TGC host by cultivating in vitro in a culturemedium is used to prepare it for carrying out the TGC process in theselected host organism. The TGC host strain can alternatively also bepropagated in the host organism (human or animal, denoted as TGC host),by in vivo cultivation. In preparation for infection, the TGC hoststrain is suspended in a non-bactericidal solution adapted for the TGChost, in a buffer or in another physiological liquid. The liquid isadministered to the TGC host, for example to the lactating mammal if theudder is to be made somatically transgenic. This can be carried outperorally by drinking the liquid or by supplying it via a stomach tube,the anus or another body orifice. The administration of the TGC hoststrain by injection is an alternative possibility and can be doneintravenously, intramuscularly directly into the target organ or,preferably, intraperitoneally. A further alternative is infecting byproducing an aerosol and then inhaling the droplets.

The TGC host (human or farm animal: cow, horse, goat, sheep, pig, hare,poultry etc.) can be infected several times with the same orheterologous transgenes. By repeated infection with different DNA which,for example, code for several enzymes of a biosynthetic pathway, wholeenzyme cascades can be established in the TGC host. The biochemicalexpression of multigenic proteins can thus also be achieved.

e) Organ and cell specificity of infection:

The subsequent path of the TGC host strain in the organism is determinedby the natural route of infection. The TGC host strain reaches thetarget organ using the route typical for the respective bacterium. Ifthe TGC host strain carries genetically unaltered internalin, as in thecase of L. monocytogenes, then the udder will be among the targetorgans. Genetically altered internalins permit the infection of otherorgan systems. Depending on its infection cycle, the TGC host strainpenetrates into the cells and appears in the cytoplasm. As it isgenetically defective, the TGC host strain cannot proliferate there andit undergoes “endogenous suicide” (see C.1 under b) above). With cellinfection the TGC host strain has introduced the host-foreign TGC DNAinto the cell. The transfer of foreign DNA into the cell can, however,also be brought about by “exogenous suicide” (see C.4 under point b)above) or by elimination the bacteria through specific antibiotictreatment (see C.3 under point b) above). In these three cases thebacteria cells carrying the foreign DNA die within the animal cells andthereby release the foreign DNA into the cytoplasm. Finally, thetransfer of the foreign DNA into animal cells can also be achieved bytargeted infection of cells with absence of lysis of the endosomes. Theforeign DNA of the animal cells is thus available within the endosomesby lysis of the bacteria.

In each of the cases mentioned, the DNA transferred into the cells isnow available as a template for the production of the desired foreignprotein. The nucleic acid can also have a direct therapeutic effecthowever, for example by the generation of anti-sense RNA. The cells,tissue or organ manipulated in this way became somatically transgenic inthe course of the infection.

f) L. monocytogenes induced protein production in the milk of mammals

After carrying out the TGC procedure—for example with TGC host strainsuch as L. monocytogenes or other intracellularly active bacteria (e.g.Yersinia) or bacteria which penetrate the endosome (e.g. Salmonella) orare “armed” with additional bacterial factors, or otherwisenon-pathogenic bacteria (e.g. Escherichia coli or L. innocua)—theprotein is created in the lactating cell and passed out into the milkwith the other products of the cell. If several animals are madesomatically transgenic with different foreign DNA in a TGC process, thenthe different proteins can be produced, separated from each other, bycollecting the milk of each single TGC host (milking).

Due to the properties of the TGC host strain, no L. monocytogenes (TGChost strain, i.e. host bacterium) appear in the milk. Should this be thecase however, then the bacteria can be eliminated using the methodsfamiliar to an expert in the field, for example by treating withantibiotics. Animals (or also humans) are free of any viable,genetically engineered organisms after carrying out targeted geneticconditioning (TGC) and do not therefore have to submit to any furthersafety checks. The TGC host transmits the genetic information introducedinto it by the TGC process to the offspring cells in the context ofusual cell division. The information is not transmitted to thedescendants of the TGC host however, as the TGC DNA is not present inthe germ line of the TGC host. The avoidance (i.e. omission) of geneticmanipulation of the germ line of the whole organism and targeted proteinproduction in a predetermined organ or tissue of the animal host (animaland human) constitutes the innovative and new aspect of the methodaccording to the invention.

g) Infections of tissue by L. monocytogenes

Blood is a tissue whose genetic alteration using the TGC methodaccording to the invention will be described as an example. Blood cellsare particularly suited for the TGC method. It is possible to infectblood cells outside the body. The desired somatic transgenesis of thecells can similarly be monitored outside the host. In the case ofattenuated auxotrophic bacteria—diaminopimelic acid is here used as anexample for auxotrophy—the substances necessary for the growth of thecells can be added to the medium and thus control the life span of thebacteria according to the experimental objective. It is possible tocheck whether the intracellular bacteria are still alive by subsequentlysis of the animal cells.

The transfected cells, containing a well defined quantity of livebacteria, are finally used for reimplanting in the recipient organism.In particular cases there can be such a large number of bacteria thatadditional organs in the organism are infected. In other casestransgenesis is specifically restricted to the blood tissue by the invitro elimination of live bacteria before reimplantation in the TGChost.

Reimplantation and the connected dissemination of transgenic cells withor without live bacteria permits somatic gene therapy of cells in thehost, which in this case may also be a human host.

The TGC method also enables extracorporal proteins to be produced. Forthis purpose TGC host strains are injected into the eggs of poultrybirds. Suitable techniques for this are state of the art in theproduction of vaccines by viral agents. During the incubation period thecells in the egg are infected in a somatic transgenic process and thenproduce the foreign protein. The foreign protein can be purified fromthe egg using state of the art techniques. With this type of TGC processthe TGC host strain remains controllable in all stages of use underlaboratory conditions. The quantity of protein to be produced dependsonly on the injection of a correspondingly large number of eggs.

h) Use of the TGC method for somatic gene therapy

There is not yet an established form of somatic gene therapy. At presentthe nucleic acid used for transfection is protected from the influenceof the outside world within viruses or packed in liposomes.

Viruses have the disadvantage that they only have a limited sizeuptakecapacity and that the development of their full cytopathic effectat high infection doses must be taken into account [32a]. They induceimmune reactions and so can be attacked and destroyed themselves. Someviruses are inactivated by serum and are then unusable for gene therapy.Here particularly, mention should be made of the multiple dosage ofviruses for gene therapy, in the course of which the immune response ofthe host is stimulated. The creation of a specific defence aimed againstviruses has proved to be a significant problem in the use of viruses inthe context of gene therapy.

When using liposomes, the toxic effect of lipids in provokinginflammatory reactions must be considered.

In the case of in vivo therapy there are still considerable obstacles tousing the gene transfer systems used so far. For this form of therapy itis necessary to have [32b]:

-   -   (i) Resistance of the vector against breakdown after in vivo        administration in the body,    -   (ii) Tissue specificity, i.e. targeted control of the tissue        (organ) being subjected to therapy and    -   (iii) Safety, by which is meant harmlessness to organs not being        treated [32b].

The bacteria described in this patent application, which function as avehicle for gene delivery are ideally suited for gene transfer. Thebacteria are optimally adapted to their corresponding host and cansurvive in it for a sufficient length of time without externalintervention, such as antibiotic therapy. They induce specific diseasesfollowing a defined route of infection and in so doing partly displaymarked organotropy. They can take up considerable quantities of foreignDNA (e.g. naturally occurring plasmids have sizes of several hundredkilobases), so not only cDNA's but even larger regions of a chromosomecan be transferred. Finally, they can be used safely, particularly if“disabled” bacteria are used, as described above. The genetic defects ofthe TGC host strain, in combination with their antibiotic sensitivity,assure efficient elimination of the bacteria after they have completedtheir task of DNA transfer into eukaryotic cells.

Example

Examples for somatic gene therapy are listed below:

Therapy for cystic fibrosis (CF): the bacterium must here beadministered by inhalation to the patient undergoing therapy. Thebacterium used should preferably be a bacterium which is transmittedthrough droplet infection. The bacterium contains the CFTR gene, whichcan cure the crucial defect occurring in CF. The bacterium penetratesinto the airway lumen-facing columnar cells and transfects them with theCFTR DNA integrated into the TGC vector. The cells become somaticallytransgenic, the defect is cured.

β-thalassaemia can be treated by somatic gene therapy with humanβ-globulin gene. Ex vivo cells that originate from the haemopoeticsystem are infected with a TGC safety strain, which transfers theβ-globulin gene into the original cell. The infecting bacterium iseliminated by treatment of the cells in the cell culture and thetransgenic cell is prepared for transfer back into the human. Thistransfer takes place through intravenous administration.

In therapy of Hurler syndrome, naive CD34 positive cells of the bonemarrow are transfected with α-L-iduronidase gene. The way gene therapyis carried out and the transfer of the cells back into the patient areas described in the preceding example.

In gene therapy of Fanconi anaemia, the gene of the Fanconi anaemiacomplementation group C (FACC) is used for somatic gene therapy. Thetarget cells of the infection with TGC host strain are again CD34positive cells of the bone marrow.

i) Proof of the Success of TGC Method

DNA transfer is already evident in mice within the first 24 hours, i.e.long before a specific immune response against the bacterium couldarise. This was demonstrated by the production of β-galactosidase or thegreen fluorescent protein (EGFP) in cell cultures within 24 hours. The“mitogenetic effect of bacteria”, which additionally occurs in thecontext of infection, favours the establishment of DNA in the TGC celland is therefore desired and advantageous for the success of the TGCprocess.

In summary, it can be established that the use of bacteria for somaticgene therapy is safer than gene therapy using viral systems. Bacterialinfection can both be directed and restricted locally. Growth and henceflorid infection by the bacteria can be prevented by removing particularbacterial factors. Additionally the growth of bacteria in eukaryoticcells can be directly influenced and generally prevented. Finally, thetermination of bacterial infection is possible at any time through theuse of antibiotics, i.e. the place, time and effectiveness of theinfection can be controlled.

The invention is described in detail below, using L. monocytogenes as anexample:

EXAMPLE 1 Production of TGC Safety Strains

The L. monocytogenes safety strains are produced by targeted geneticalterations of primary pathogenic L. monocytogenes. In so doing, severallevels of safety are established together. Recurrence of vitality orpathogenicity caused by reversion of the mutations is prevented. Themutations affect genes which (1) influence the survival of bacteria inthe cell, (2) which diminish the pathogenicity of the bacteria in theTGC host and (3) which prevent survival of the bacteria in theenvironment, should any escape.

a) First level of safety—safety relevant property: survival in theenvironment (see point B.1 under b) above)

TGC host strain s can be applied to the TGC host either by injection orby peroral administration. With peroral administration there may be asurplus of bacteria, resulting in secretion of bacteria, which are notingested by the organism. In order that these eliminated bacteria haveno opportunity of surviving in the environment, the TGC safety straincan contain additional mutations which prevent the growth of thebacteria in the environment.

As an example for this, the switching off of the cspL gene (cold shockprotein of Listeria) is indicated. This has the consequence that thebacteria can no longer grow at temperatures under 20° C. Growth andability to infect at 37° C. are not adversely affected, but areadditionally modulated by simultaneous mutations according to a) and b).The cspL gene, which is deleted in the safety strains used in thisinvention, is shown in the sequence protocol under SEQ. ID No. 2. Acorresponding cspL deleted strain has been deposited at the DSM underNo. 11883 with the description L. monocytogenes EGD delta cspL1.

The TGC safety strains of the invention can only be cultivated onspecial growth substrates. The growth temperature must be above 37° C.,growth is not possible below 20° C. The bacteria possess limitedpathogenicity and are only capable of penetrating restricted, tightlydefined areas of the TGC host. In this way safety of the system forhumans and the environment is assured. The TGC host strains are nolonger able to grow outside the artificial media, here specifically, thehost cell. This restricted intracellular viability is at the same time aprerequisite for the release of TGC DNA in the host cell and hence forthe induction of somatic transgenesis using the TGC method.

b) Second level of safety—attenuation: reduced pathogenicity (see pointB.2 under b) above)

The second level, of attenuation of the TGC safety strains includesmutations in the pathogenicity factors. Through targeted mutations indefined factors, pathogenicity in the bacteria is reduced, inducedapoptosis of infected host cells is prevented and the immune reaction isat the same time directed in the desired direction. The mutationsrestrict the intracellular motility of the bacteria and hence theirspread to secondary cells. The infection is thus limited to the chosentarget cells, with retention of treatment using antibiotics.

For safety considerations it is desirable to restrict or even preventthe intracellular spread of TGC nurse after infection. Accurateknowledge of the intracellular lifestyle and the motility of the abovementioned bacteria makes it possible to produce defined, stable mutantswith reduced ability to infect the TGC host.

With L. monocytogenes, the mutations attenuated in this way affect, forexample, the hly gene with consequent blocking of infection in the firstcell. An example for the switching off of this pathogenicity factor, thestrain L. monocytogenes EGD HlY_(D491A) has been deposited and hasreceived the number DSM 11881.

Another example for the reduction of pathogenicity of L. monocytogenesare mutations in actA gene or the deletion of regions which arenecessary for the interaction between actA and the host cell proteinVASP, with the consequent blocking of intracellular motility. Finally,there are mutations of plcB gene, in which bacteria are disabled forspread into a second cell. The deposited strain L. monocytogenes EGDdelta actA delta plcB is an example of a double mutation in which boththe actA gene and the plcB gene are removed. It has deposit number DSM11882.

It is additionally possible to exchange the wild-type listeriolysin genein L. monocytogenes for a mutated allele. The properties of thelisteriolysin are then restricted, both for inducting apoptosis invarious host cells and also for generating a strong T cell mediatedimmune response.

C) Survival in the cell:—endogenous suicide: third level of safety (seepoint C.1 under b) above)

In general one of the features of attenuated bacteria for the TGCprocess is their having defined deletions in the genes which areessential for the biosynthesis of integral bacterial components. Theselected auxotrophic bacteria are suitable as TGC host strains, since,being attenuated bacteria, they can transport foreign DNA into the cell.However, as the bacteria in the cells lack essential “growth factors”,they spontaneously lyse and thereby release TGC DNA in the cell.

L. monocytogenes are used as TGC safety strains. They are geneticallyaltered in such a way that although they infect the cell, they can nolonger multiply in the cell. This is achieved by, for example,inactivating the dapE gene in L. monocytogenes. Listeria are grampositive bacteria which, just like gram negative bacteria, requiremeso-diaminopimelic acid derivative (DAP) for cross-linking of the cellwall. Biosynthesis of diaminopimelic acid is therefore essential for thecreation of the bacterial cell wall. DAP auxotrophic bacteria succumb tospontaneous lysis if this amino acid is no longer supplied in theculture medium. The enzymes which are involved in DAP synthesis inbacteria are not present in mammalian cells. In TGC safety bacterialstrains, these enzymes are also deleted or inactivated by insertions orother means. The dapE of L. monocytogenes, which was inactivated in thesafety strains used according to the invention, is shown in the sequenceprotocol as SEQ. ID No. 1. For the genetic manipulation of the dapE genein L. monocytogenes, its sequence had to be determined, as correspondinggenes, e.g. from E. coli, has only about 30% homology to the sequence ofSEQ ID No. 1 protocol.

The bacteria deleted for this or other genes of the DAP biosynthesispathway, so called DAP mutants, cannot grow either within or outside thehost. In order to grow they require the addition of a large quantity ofDAP (1 mM) to the growth medium. If DAP is missing, the bacterium cannotsurvive either in the TGC host or outside the TGC host. These DAPmutants hence provide safety, both against a bacterial infection of theTGC host and safety against an infection of other organisms in case ofrelease of a strain of this type into the environment.

A manipulation of the genome of Salmonella (creation of an auxotrophicmutant) shows that the deletion (or blocking or mutagenesis) of the aroAgene, which is essential for the synthesis of aromatic amino acids, hasthe same effect. From the Salmonella vaccine strain (available from theAmerican collection of bacterial strains under the number ATCC14028), amutant can be produced by genetic manipulation using techniqueswell-known to experts, and with knowledge of the aroA gene sequence(Genebank accession number M10947). This mutant can function as a TGCsafety strain in a similar way to the recombinant bacteria heredescribed for Listeria. Release of foreign DNA occurs, as for the abovedescribed L. monocytogenes delta dapE strain, through the bacteria dyingoff after their uptake into the cell. Unlike L. monocytogenes,Salmonella cannot enter the cell cytoplasm. Release of the foreign DNAin this case occurs from the endosomes into the cell cytosol.

Other attenuated mutations of L. monocytogenes are also known, in whichbiosynthesis of nucleic acids, amino acids, sugars or other essentialcell wall ingredients, is blocked [33 to 35]. The same can also beachieved through mutations in regulatory genes which are essential forthe intracellular lifestyle of the bacteria. An example of a gene ofthis type is phoP of Salmonella typhimurium [36].

The examples described here for L. monocytogenes can be applied to otherintracellular live bacteria or bacteria which are first made intointracellular activators by being armed with pathogenicity factors. Thisis especially the case for bacteria of the types Aeromonads, Bartonella,Brucella, Campylobacter, Clostridia, Enterobacteriaceae (particularly E.coli, Salmonella, Shigella, Yersinia), Mycobacterium, Renibacterium andRhodococcus. A TGC safety strain accordingly armed, for example,Bacillus subtilis, which is additionally equipped with listeriolysinfrom L. monocytogenes.

An important prerequisite for transfer of DNA itself into cells distalin the body is the protection of the DNA on its way to the target cellor target tissue or target organ. The ability of intracellular livebacteria such as L. monocytogenes to spread intracellularly is an idealproperty for transporting genes into isolated cells, deeper tissue andorgans. The vehicle, the TGC host strain, dies after successful transferof TGC DNA into the target cell, as a consequence of attenuation (B.1),induction of auxotrophy (B.2), endogenous suicide (C.1), infection byendosomes (C.2), infection by phagolysosomes (C.3), exogenous suicide(C.4) or antibiotic therapy (D.1).

EXAMPLE 2 Release of Foreign DNA in Animal Cells (Tissue or Organ)

A) Infection via endosomes: Transfer of the expression plasmid withoutrelease of the bacteria from the endosome vesicle (see point C.2 underb) above)

Tests were carried out to see if bacteria are able to transfer theirplasmid DNA into the cytoplasm of infected host cells, without it beingnecessary for them to first escape from the endosome vesicle. Theability of L. monocytogenes Δhly mutants, which can no longer leave theendosome, to function as a transfer bacterium for DNA transfer wasinvestigated. EGFP was chosen as the foreign DNA to be transferred. Itis a fluorescent protein which was cloned under the control of a CMVpromoter. As a measure for successful transfer of foreign DNA—i.e. as ameasure for transfection of the eukaryotic cells—10,000 cells wereexamined in a FACS scanner for the occurrence of EGFP dependentfluorescence, after infection with the corresponding L. monocytogenesstrains. The number is expressed in Table 1 as a percentage of the totalnumber of measured eukaryotic cells. L. monocytogenes wild-type strainEGD served as a positive control during the experiments. An isogenicnon-invasive ΔInlAB strain was also tested. The evidence obtained withthese bacteria have general validity and are transferable to otherbacteria.

The results are summarized in Table 1 and show that Δhly mutant is justas efficient as the wild-type L. monocytogenes strain with regard to DNAtransfer from the bacterium into the eukaryotic cell. The L.monocytogenes ΔInlAB strain is not suitable (PtK2) or is significantlyworse (Hep-2) as a vehicle for DNA transfer into the cells hereindicated. The experiments also show that the active uptake of bacteriaby eukaryotic cells (in this case non-professional phagocytes) is aprecondition for transfection of cells. The attachment of bacteria iseffected by the interaction between bacterial internalins (InlA and/ orInlB) and the receptors of the animal cells. The experiments of thefollowing example demonstrate that internalin is not necessary for theuptake of bacteria in professional phagocytes. Cell L. monocytogenesTransfected line Origin strain cells in % PtK2 Kangaroo rat kidneyWild-type EGHD 1.71 Δhly 1.78 ΔinlAB 0 Hep-2 Human larynx Wild-type EGHD4.58 carcinoma Δhly 4.31 ΔinlAB 0.24

b) Infection through phagolysosomes: Arming of non-pathogenic strains asTGC safety strain; (see point C.3 under b) above)

The example shown below for L. innocua is representative and can beextended to other non-pathogenic bacteria (e.g. Escherichia coli). Thesteps leading to the genetic manipulation of such bacteria correspond tothose here indicated for L. innocua.

A non-pathogenic L. innocua strain (Serovar 6a) was “armed” with thepathogenicity factors listeriolysin and ActA from listeriamonocytogenes. In order to be able to regulate this gene, thepositive-regulatory factor (PrfA) was cloned as third gene intogenetically engineered L. innocua strain. The presence of PrfA causesexpression of the virulence gene to be growth temperature dependent. Asthis recombinant L. innocua strain possesses no internalin, i.e. is notitself invasive, it cannot penetrate into the above mentioned cells(Ptk2, Hep-2). If the experimenter wishes to be able to also infectthese cells, then the bacteria must additionally be equipped with theinternalins InlA and/ or InlB. The experiments of the present exampleshow that there is no need of these bacterial products (internalins) forthe ingestion of L. innocua (hly+; actA+) strain by professionalphagocytes. After their phagocytosis, the L. innocua strain (hly+;actA+) uses the protein listeriolysin for the lysis of thephagolysosomes of the professional phagocytes. It can be seen from theelectron micrographs that the genetically manipulated L. innocua (hly+;actA+) strain appears in the cytoplasm of the professional phagocytes.The wild-type strain L. innocua Serovar 6a, on the other hand, is killedoff in the phagolysosome and does not appear in the cell cytoplasm.Expression of the ActA-protein enables the L. innocua (hly+; actA+)strain to have an actin cytoskeletal-dependent intracellular movement,which appears similar to the movement of the L. monocytogenes strains inthe EM images. Due to the failure of further genes, such as e.g. theplcB gene, the L. innocua (hly+; actA+) strain mentioned here cannotspread to neighbouring cells. This specific alteration in infectivityhas already been described for recombinant L. monocytogenes AplcBstrains.

The targeted selection of genes, here hly and actA, and theirtransformation into non-pathogenic bacteria, transfers the selected L.monocytogenes properties to non-pathogenic bacteria. The escape of thebacteria from the “deadly” phagolysosome is a precondition for thetransfer of foreign DNA into infected cells. The DNA which is to betransferred for the reprogramming of animal cells, is thereby integratedinto host strains, as described above for attenuated L. monocytogenesbacteria—which according to the invention can be used as such. Therelease of the genetic information according to the invention occursthrough (i) creation of auxogenous mutants (deletion of endogenous,life-essential genes), (ii) through introduction of “suicide genes”,(iii) through induced ingestion into endosomes and killing off there or(iv) through antibiotic therapy which is temporally defined and directedto killing bacteria in a target organ or tissue.

The experiments of this example are representative of how naturallyoccuring non-pathogenic bacteria can be consecutively “armed”. Byequipping them with defined bacterial factors (here genetic i.e.properties of naturally invasive bacteria), bacteria which are otherwiseprimarily unsuited for the TGC method can be manipulated and directed insuch a way by the experimenter so that they can be used for controlledinfection and transfer of DNA into animal cells (or tissue, organ, wholeanimal, human).

c) Release through exogenous suicide: Cloning of suicide genes: (seepoint C.4 under (b) above)

Suicide genes, which are activated after penetrating into the host celland lead to death of the bacteria, can be supplied to the bacteria inthe form of lysis genes from bacteriophages, for example with the S-geneof the lamda or analogous bacteriophages [37], or with killer genes fromplasmids [38]. These genes are controlled by an intracellular inducablepromoter (for example pagC-promoter from Salmonella [38]).

d) Release through antibiotic therapy: Targeted release of foreign DNAin the lung after droplet inhalation of Listeria monocytogenes (seepoint D.1 under (b) above).

Infection with bacteria took place according to the method “Bodyplethysmography in spontaneously breathing mice” by R. Vijayaraghavan[Arch. Toxicol. 67: 478-490 (1993)]. In the experiment mice were exposedsingly for half an hour in an inhalation chamber to an aerosol of onemillilitre of bacterial suspension, which contained a total of 5000bacteria. This quantity of bacteria corresponds to the LD50 dose ofintraperitoneally administered bacteria. In order to be able to followthe course of the infection in real time, the bacteria were once moretransformed with a EGFP-gene construct. Using fluorescence analysis ofthe EGFP-protein formed in the tissue, the route of infection of thebacteria in the animal model was followed. Within half an hour thebacteria penetrate into the columnar and endothelial cells of the airpassage. At this point no bacteria are to be found in other tissue ororgans of the infected animal, such as e.g. spleen, liver, brain. Theinfection remains exclusively restricted to the lung for up to 18 hours.Only after 24 hours are other organs also affected.

The experiment shows that the spread of bacteria after droplet infectioncan be restricted to the primary organ if there is an intervention intotheir viability. Two ways of achieving this are by using attenuatedmutants (e.g. ActA deleted in the “spreading gene”) and/or by destroyingthe bacteria through initiating antibiotic therapy at a time determinedby the experimenter, i.e. in an organ determined by the experimenter.

EXAMPLE 3 Description of the TGC Vectors

TGC vectors are episomal DNA, for example plasmids with low ingestioncapability for foreign DNA (pMB derivatives which are sufficient forsingle genes), or plasmids with greater DNA ingestion capability (suchas in Pl- or F-plasmids), in order to create somatic transgenesis forcomplex biosynthetic pathways.

In all cases, the plasmids involved are replicated in the bacteria hostswhich are used for genetic alteration and cultivation for the TGCprocess. E. coli, or other bacteria commonly used in recombinant DNAtechniques, are suited as examples of an intermediate host in whichgenetic building blocks can be constructed. L. monocytogenes or otherabove-mentioned bacteria functioning as TGC host strainss are suitableas a TGC safety strain. In order to fulfil this condition, the plasmidscontain the host-specific plasmid replicon sequences. During the processof generating recombinant DNA, the transformed host cells must bedistinguished from “naked” host cells. Generally, common antibioticresistance genes can be used as selection principles for this.

EXAMPLE 4 Transformation of L. monocytogenes Safety Strains to TGC HostStrains

The transformation of L. monocytogenes is carried out according to amodified protocol of Park and Stewart [40].

Accordingly, bacteria are applied up to an optical density of OD₆₀₀=0.2.Ampicillin (10 μg/ml) and 1 mM glycine are added to the culture medium.Further proliferation occurs up to an OD₆₀₀ of 0.8 to 1.0. The cells areharvested by centrifugation and resuspended in 1/250 vol. coldelectroporation buffer (1 mM Hepes, pH 7.10, 0.5 M sucrose). Thebacteria are washed up to four times prior to electroporation.

For electroporation, 50 μl of the prepared cells are added to anelectroporation cuvette, electroporation is carried out using 1 μg DNAat 10 kV/cm, 400 ohms, 25 μF.

After electroporation the cells are immediately cooled on ice, suspendedin 10× BHI medium and incubated for 2 hours at 37° C. with carefulagitation. After this the cells are plated and incubated at the desiredtemperature. The efficiency of transformation with this method is 10⁴ to10⁵ transformers per μg plasmid DNA used.

EXAMPLE 5 Description of the Cultivation of TGC Host Strains For Use inthe TGC Method

Listeria were preferably cultivated in the brain-heart infusion broth,for example BHI of the Difco company. Alternatively, and for specialapplications (radioactive labelling of listerial proteins), the bacteriacan be cultivated in tryptic soy broth (TSB) or in Listeria minimalmedium (LMM) [36]. The bacteria are centrifuged off and washed severaltimes in a suitable transfer medium, for example, a bicarbonatecontaining buffer.

Bacteria prepared in this way can be kept for at least 6 months at −80°C. with the addition of 15% glycerine solution, before they are used inthe TGC procedure.

EXAMPLE 6 TGC Method—Use of TGC Host Strains as Nutrient

As an introduction to the TGC process, the animals are not allowed todrink for a few hours. The (TGC host strain: TGC-DNA in the desiredstrain) are infused in a bicarbonate containing buffer of suitableconcentration and administered to the animals orally, by inhalation orby injection (parenteral, intramuscular, intraperitoneal or directlyinto the target organ). The type of application is determined by thephysiological route of infection of the corresponding TGC hot strain.The selection of the bacterium which is used as TGC safety straindepends on the target organ and is established according to the path ofinfection and according to the organotropy of the relevant bacterium.The dosage of bacteria is chosen so as to achieve the desiredorganotropic transfection of the TGC host strain. The quantity and typeof bacterial application thus depends on the particular bacterium, butalso depends on the host and target organ (see also example 2).

EXAMPLE 7 Implementation of Somatic Gene Therapy

Examples for somatic gene therapy are listed below:

Therapy for cystic fibrosis (CF): the bacterium must be administered byinhalation to the patient undergoing therapy. The host used shouldpreferably be a bacterium which is transmitted through dropletinfection. The bacterium contains the CFTR gene, which can cure thecrucial defect occurring in CF. The bacterium penetrates into the airwaylumen-facing columnar cells and transfects them with the CFTR DNAintegrated into the TGC vector. The cells become somatically transgenic,the defect is cured.

β-thalassaemia can be treated by somatic gene therapy with humanβ-globulin gene. Ex vivo haematopoetic stem cells are infected with aTGC safety strain, which transfers the β-globulin gene into the originalcell. The infecting bacterium is eliminated by treatment of the bacteriain the cell culture and the transgenic cell is prepared for transferback into the human. This transfer takes place through intravenousadministration.

In therapy of Hurler syndrome, primitive CD34 positive cells of the bonemarrow are transfected with α-L-iduronidase gene. The way gene therapyis carried out and the transfer of the cells back into the patient areas described in the preceding example.

In gene therapy of Fanconi anaemia, the gene of the Fanconi anaemiacomplementation group C (FACC) is used for somatic gene therapy. Thetarget cells of the infection with TGC host strain are again CD34positive cells of the bone marrow.

EXAMPLE 8 Monitoring the Success of Induced Somatic Transgenesis

After the TGC DNA has been transferred into the TGC host, the success ofthe TGC process has to be monitored. Immunological methods for detectinggene products (proteins) are suited for this, such as immunoassays (e.g.ELISA), immunoblot or other well-known methods which involve anantigen-antibody reaction. T-cell responses can be measured in specialassays and are always used when the antigen is a substance that isrecognized via MHC-class 1 mediated immune responses.

If the protein produced is an enzyme, then its biological activity canbe determined in the form of an enzyme activity test. If the proteinadditionally possesses biological activity, then the efficiency of theprotein produced can be measured with biological assays.

For proteins that induce passive or active immunisation of the TGC host,protection against the activating agent can be tested; for example, theprevention of colonisation, infection (or apparent disease) in theexperimental animal after exposure to the pathogenic organism (bacteriumor virus).

EXAMPLE 9 Harvesting the Protein

The protein to be produced can be obtained using state of the arttechniques that are common knowledge to persons involved in animalhusbandry:

if the TGC host is a cow or other lactating farm animal and the udder isthe infected organ, then the well-known techniques of milking can beused;

if poultry birds such as hens were used as the TGC host, then the eggsare collected and taken to the protein purification stage;

processing of proteins from organs whose products cannot be externallyaccessed is achieved by obtaining the relevant organs, for which theanimal must usually be killed, e.g. with fish;

if the somatic transgenic tissue is blood, then the desired product isobtained after venous aspiration, from the blood or its cells andpurified by methods familiar to the expert.

EXAMPLE 10 Initial Purification of the Protein

Preliminary purification of the protein to be produced is achieved byseparation processes, which are familiar to the expert as mainlyphysical or physico-chemical methods. Amongst these are precipitatingthe proteins using salts (for example, ammonium sulphate), acids (forexample, trichloroacetic acid) and using heat or cold.

A rough separation can also be achieved via column chromatography. Allthe methods used here strongly depend on the primary media in which theprotein is enriched. For example, many methods are known for theprocessing of milk or eggs in industry, and they can be used in theinvention described here. The same also applies to processing of bloodas a somatic transgenic tissue. Here it is possible to refer to theexperience of transfusion medicine, particularly the processing andpurification of blood clotting factors.

EXAMPLE 11 Purification of the Protein

For the final purification of the proteins, all the methods used inconventional purification of proteins can be used. Amongst them are:

purification using affinity chromatography, for example exploiting thereceptor-ligand interaction;

the preparation of fusion proteins with so-called “tags”, which can beused for specific interaction with a matrix in chromatography (forexample, polyhistidine tag and nickel column chromatography; thestreptavidin-biotin technology of affinity purification). The tags canbe then removed by appropriate introduction of a corresponding proteasecutting site allowing subsequent release of the desired proteinfollowing protease digestion;

purification via specific antibodies (immunoaffinity chromatography);

the exploitation of natural affinities between the target protein andother proteins, carbohydrates or other binding partners, as in the caseof toxin A of Clostridium difficile, which binds to thyroglobin at 4° C.and is subsequently eluted by raising the temperature to 37° C.

EXAMPLE 12 Production of TGC Proteins

The list of proteins which it is possible to produce with the TGC methodis theoretically unlimited and above all includes the range of hormones,regulatory factors, enzymes, enzyme inhibitors and human monoclonalantibodies, as well as the production of surface proteins of pathogenicmicroorganisms or viral envelope proteins so as to safely producediagnostic tests and vaccines which can be tolerated. The list covershigh volume products such as human serum albumin and also proteins usedin smaller quantities, such as hirudin, blood clotting factors, antigensfor tumour prophylaxis and for active immunisation (for example,papilloma antigen) or for passive immunisation.

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1-22. (Canceled)
 23. A bacterium useful as a vehicle for gene transportand gene transfer to eukaryotic cells of specific organs or tissues ofan organism comprising: a foreign DNA integrated into an episomalvector, wherein the transcription and expression of the foreign DNA isunder the control of a eukaryotic regulatory sequence, wherein thebacterium: a. is vital and viable in the organism; b. exhibit one ormore properties chosen from: i. fully pathogenic; ii. attenuated toprevent the bacterium from inducing death of the eukaryotic cells, iii.attenuated to restrict the intracellular motility of the bacterium, iv.attenuated to permit efficient elimination after transfer of the foreignDNA to the eukaryotic cells; v. non-pathogenic; and vi. naturallynon-pathogenic and provided with additional pathogenicity factors, saidfactors enabling the bacteria to infect the organism in a controlledmanner, to advance into the organs and tissues of the organism, and totransfer the foreign DNA to remote somatic cells; c. can infect thespecific eukaryotic cells via its typical natural route of infection andcycle of infection; d. has the route of infection that is directed andlocally limited either naturally or due to a specific genetic alterationof one or more genes; and e. has an infection cycle that can be limitedin time and terminated either by use of an antibiotic or by use ofauxothropic mutants of the bacterium.
 24. The bacterium of claim 23,wherein the eukaryotic regulatory sequence originates from a previouslyselected target organ or is optimized for the target organ.
 25. Thebacterium of claim 23, wherein the bacterium further comprises anadditional exogenous suicide gene.
 26. The bacterium of claim 23,wherein the one or more genes in step d are chosen from: i. genes thatinfluence the reproduction of the bacterium in the eukaryotic cells, ii.genes that reduce the pathogenicity of the bacterium in the organism,and iii. genes that inhibit the survival of the bacterium in theenvironment after the bacterium is excreted from the organism.
 27. Amethod for the production and extraction of proteins comprising: a)providing a bacterium useful as a vehicle for gene transport and genetransfer to eukaryotic cells of specific organs or tissues of anorganism (a TGC procedure) comprising a foreign DNA integrated into anepisomal vector, wherein the transcription and expression of the foreignDNA is under the control of a eukaryotic regulatory sequence; b)infecting the eukaryotic somatic cells of the organism with thebacterium to produce transgenic cells, said transgenic cells expressingthe foreign DNA to produce a foreign protein encoded by said foreignDNA; and c) isolating the foreign protein from the cell, tissue ororgan, wherein the bacterium:
 1. is vital and viable in the organism; 2.exhibit one or more properties chosen from: i. fully pathogenic; ii.attenuated to prevent the bacterium from inducing apoptosis of theeukaryotic cells, iii. attenuated to restrict the intracellular motilityof the bacterium, iv. attenuated to permit efficient elimination aftertransfer of the foreign DNA to the eukaryotic cells; or v.non-pathogenic; and vi. naturally non-pathogenic and provided withadditional pathogenicity factors, said factors enabling the bacteria toinfect the organism in a controlled manner, to advance into the organsand tissues of the organism, and to transfer the foreign DNA to remotesomatic cells;
 3. can infect the specific eukaryotic cells via itstypical natural route of infection and cycle of infection;
 4. have theroute of infection that is directed and locally limited either naturallyor due to a specific genetic alteration of one or more genes selectedfrom the group consisting of: i. genes that influence the reproductionof the bacteria in the eukaryotic cells, ii. genes that reduce thepathogenicity of the bacteria in the organism, and iii. genes thatinhibit the survival of the bacteria in the environment after thebacteria is excreted from the organism; and
 5. has the route and cycleof infection that can be limited in time and terminated either by use ofan antibiotic or by use of an auxothropic mutant of the bacterium. 28.The method of claim 27, wherein the eukaryotic regulatory sequenceoriginates from a previously selected target organ or is optimized forthe target organ.
 29. The method of claim 27, wherein the bacteriumfurther comprises an additional exogenous suicide gene.
 30. The methodof claim 27, wherein the bacteria is chosen from a genus selected fromthe group consisting of: Aeromonas, Bartonella, Brucella, Campylobacter,Clostridia, Enterobacteriaceae, Legionella, Listeria, Mycobacterium,Renibacterium, Rhodococcus, and a genus that is genetically orbiochemically related to them.
 31. The method of claim 27, wherein thebacterium belongs to the genus Listeria.
 32. The method of claim 27,wherein the bacterium is of strain Listeria monocytogenes.
 33. Themethod of claim 27, wherein the bacteria is of the strain Listeriamonocytogenes EGH HlyD_(491A), which is deposited at the DSMZ (Germancollection of Microorganisms and Cell Cultures) under the number 11881.34. The method of claim 27, wherein the bacteria is of the strainListeria monocytogenes and EGD Delta actA Delta plcB, which is depositedat the DSMZ (German collection of Microorganisms and Cell Cultures)under the number of
 11882. 35. The method of claim 27, wherein thebacteria is of the strain Listeria monocytogenes EGD Delta cspL 1, whichis deposited at the DSMZ (German collection of Microorganisms and CellCultures) under the number of
 11883. 36. The method of claim 27, whereinthe organism is selected from the group consisting of: (a) a domesticanimal, with the transgenesis being induced in the blood or other tissueof the working animal, (b) a lactating animal, with the transgenesisbeing induced in the udder of the lactating animal, and (c) poultry,with the transgenesis being induced in eggs of the poultry.
 37. Asomatic transgenic domestic animal produced by the method of claim 27.38. The method of claim 27 in which the somatic transgenic tissuecreated through infection with the bacterium of claim 1 is reimplantedin an entire organism.
 39. The method of claim 27 wherein the foreignprotein is selected from the group consisting of hormone, regulationfactor, enzyme, enzyme inhibitor, and a human monoclonal antibody. 40.The method of claim 27 further comprising using the foreign protein as adrug, vaccine, or for preparation of diagnostics.